The epithelium of mammalian lungs are lined with an endogenous pulmonary surfactant (PS) which facilitates breathing by aiding the transport of oxygen across the lung air-liquid interface. A deficiency in this surfactant is the primary cause of neonatal respiratory distress syndrome (RDS) and is linked to RDS in adults as well. Native PS is a mixture of lipids and proteins, and although its exact composition is unknown, researchers have prepared a number of exogenous surfactants which are useful in the treatment of RDS in pre-term infants. KL-4 is an example of an exogenous surfactant which is useful in the treatment of RDS as disclosed in U.S. Pat. Nos. 5,164,369, 5,260,273 and 5,407,914, hereby incorporated by reference.
KL-4 is a mixture of a pharmaceutically acceptable phospholipid and a 21 residue polypeptide, (L-lysine-(L-leucine).sub.4).sub.4 L-lysine. (SEQ. ID No. 1) As disclosed in the aforementioned patents, this peptide was prepared by solid phase synthesis and recombinant DNA techniques. The solid phase synthesis comprises sequential addition of one or more amino acid residues coupled with suitable protection of amino or carboxyl groups. Although this process is effective, it is not amenable to the large scale synthesis necessary to manufacture a drug substance.
The object of the present invention is the production of the peptide component of KL.sub.4, namely (Lys-Leu.sub.4).sub.4 Lys (SEQ. ID No. 1) by a liquid phase peptide synthesis ("LPPS"). Unlike the methods disclosed in the aforementioned patents, this process is amenable to large scale synthesis.
An additional embodiment of the invention concerns the deprotection of the carboxy terminus of a peptide which was protected as an ester. Most peptide syntheses require manipulation of carboxyl and amino protecting groups. Typically, terminal carboxyl groups are protected as their ester derivative. However deprotection methods may be accompanied by racemization of the .alpha.-carbon; a problem that is compounded as the length of the peptide increases. In several steps of the synthesis of (Lys-Leu.sub.4).sub.4 Lys, an ester protected carboxyl group is deprotected. As with most biomimetic products, the configuration of the peptide is crucial and the active configuration of the residues of (Lys-Leu.sub.4).sub.4 Lys is "L". This invention discloses a method of deprotecting a peptide's ester protected carboxyl group which reduces the amount of racemized product. Although this method is applied in the synthesis of (Lys-Leu.sub.4).sub.4 Lys, it may be used in the synthesis of other peptides as illustrated hereinafter.